Ann Surg Oncol DOI 10.1245/s10434-013-3452-1
ORIGINAL ARTICLE – TRANSLATIONAL RESEARCH AND BIOMARKERS
Prevalence, Clinicopathologic Characteristics, and Molecular Associations of EGFR Exon 20 Insertion Mutations in East Asian Patients with Lung Adenocarcinoma Yunjian Pan, MD1,2, Yang Zhang, MD1,2, Yuan Li, MD2,3, Haichuan Hu, MD1,2, Lei Wang, MD1,2, Hang Li, MD1,2, Rui Wang, MD1,2, Ting Ye, MD1,2, Xiaoyang Luo, MD1,2, Yiliang Zhang, MD1,2, Bin Li, MD1,2, Deng Cai, MD1,2, Lei Shen, MD2,3, Yihua Sun, MD1,2, and Haiquan Chen, MD1,2 Department of Thoracic Surgery, Fudan University Shanghai Cancer Center, Shanghai, China; 2Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China; 3Department of Pathology, Fudan University Shanghai Cancer Center, Shanghai, China 1
ABSTRACT Purpose. To define the prevalence, clinicopathologic characteristics and molecular associations of epidermal growth factor receptor (EGFR) exon 20 insertion mutations in East Asian lung adenocarcinoma patients. Methods. A total of 1,086 lung adenocarcinomas were sequenced for EGFR mutations. EGFR and HER2 copy number variations; total and phosphorylated (p) protein expression of ErbB family members including EGFR, HER2, and HER3; phosphorylated protein expression of downstream signaling molecules including Akt and Erk; and clinicopathologic features in lung adenocarcinomas with EGFR exon 20 insertion mutations were all investigated. Results. EGFR exon 20 insertion mutations were present in 2.9 % of lung adenocarcinomas and 4.7 % of all the EGFR mutations. Compared to those with classic activating EGFR mutations, lung adenocarcinomas with exon 20
insertion mutations were characterized by significantly younger age at diagnosis (P = 0.032 for exon 20 insertions vs. L858R) and shorter relapse-free survival [P = 0.045 for exon 20 insertions versus (vs) exon 19 deletions]. Molecularly, samples harboring exon 20 insertion mutations had lower expression of phosphorylated (p)-EGFR (P \ 0.001) and HER3 (P = 0.016). In addition, higher expression of p-Akt (P = 0.007) and lower expression of p-Erk (P = 0.009) were observed in tumors with exon 20 insertion mutations. Conclusions. Lung adenocarcinomas with EGFR exon 20 insertion mutations were present in a substantial proportion. This subset showed distinct clinicopathologic features, less dependence on EGFR molecularly, and different pathway activation patterns compared to those with classic EGFR activating mutations.
INTRODUCTION
Yunjian Pan and Yang Zhang have contributed equally to this work and should both be considered first co-authors.
Electronic supplementary material The online version of this article (doi:10.1245/s10434-013-3452-1) contains supplementary material, which is available to authorized users. Ó Society of Surgical Oncology 2014 First Received: 18 August 2013 Y. Sun, MD e-mail: [email protected] H. Chen, MD e-mail: [email protected]
Lung cancer is the most prevalent cancer in male patients and the fourth most commonly diagnosed cancer among female patients.1 Non-small cell lung cancer (NSCLC) is now a disease that can be classified into clinically relevant molecular subgroups, each with distinct clinicopathologic features and potential for targeted therapies. Among these molecular subsets, the identification of epidermal growth factor receptor (EGFR) tyrosine kinase domain mutations that predict sensitivity to tyrosine kinase inhibitors (TKIs) is the most prominent event.2–4 Patients with EGFR mutations, however, are also a heterogeneous group with different sensitivity to TKIs. Although the classic exon 19 deletions and L858R point
Y. Pan et al.
mutation in exon 21 usually confer sensitivity to reversible TKIs, gefitinib, and erlotinib, most exon 20 insertion mutations reported to date are associated with resistance to TKIs.5–7 Exon 20 insertion mutations can also be classified into two subgroups by aminoacid position: insertions that affect the amino acids after the C-helix tyrosine kinase domain (Ala767 to Cys775) of the EGFR and within the C-helix (Glu762 to Met766) of the EGFR. The former account for the majority (90 %) of all reported exon 20 insertions and show de novo resistance to clinically achievable doses of gefitinib and erlotinib,5 although the latter accounts for 4 % and may be labeled EGFR TKIsensitive mutations.8 The mechanisms underlying the differential sensitivity to EGFR TKIs between EGFR exon 20 insertion mutations and classic activating mutations are largely unexplored. Despite EGFR tyrosine kinase domain mutations, other EGFR gene status including increased copy number and protein expression were reported to predict response to EGFR TKIs.9–11 EGFR (ErbB1) is a member of the ErbB family. Other ErbB family members including HER2 (ErbB2) and HER3 (ErbB3) are also important in the development and progression of human cancer.12 Ligand binding induces homodimerization and heterodimerization of the ErbB receptors, leading to the autophosphorylation of their cytoplasmic domains and the consequent activation of downstream proteins.13 Accordingly, some studies reported a correlation between the expression of the HER2 and HER3, and the therapeutic efficacy of EGFR TKIs.14–16 Moreover, because EGFR activity is mediated by downstream signaling cascade, including the PI3 K/Akt and Ras/Raf/Erk pathways, the possible roles of phosphorylated (p)-Akt and p-Erk as predictive markers have also been reported.17,18 We hypothesized that there may be differential expression of these molecular predictors between EGFR exon 20 insertion mutations and classic activating mutations, which may provide some explanation to the differential sensitivity to EGFR TKIs. We, therefore, sequenced more than 1,000 lung adenocarcinomas to investigate the clinicopathologic features of patients harboring EGFR exon 20 insertion mutations. We also explored the status of other reported predictive molecular markers for EGFR TKIs in samples harboring EGFR exon 20 insertion mutations. MATERIALS AND METHODS Patients and Samples From August 2007 to October 2012, we consecutively collected tumor tissues from lung adenocarcinoma patients who underwent surgical resection at the Department of Thoracic Surgery, Fudan University Shanghai Cancer
Center, Shanghai, China. Eligible criteria included pathologic confirmation of lung adenocarcinoma, each sample containing a minimum of 50 % of tumor cells and sufficient tissue for comprehensive mutational analyses. Patients who received neoadjuvant therapies were excluded. Clinicopathologic variables collected for analyses included age at diagnosis, gender, smoking history, tumor differentiation, pathologic TNM stage, and histologic subtypes in line with the new International Association for the Study of Lung Cancer, the American Thoracic Society, and the European Respiratory Society international multidisciplinary classification of lung adenocarcinoma.19 Disease recurrence and survival were recorded according to follow-up clinic visits or telephone calls. Our institutional review board approved this study, and written informed consent was obtained from all patients. EGFR Mutational Analysis RNA was extracted according to standard protocol from frozen tumor specimens. Total RNA samples were then reverse transcribed into complementary DNA. The most commonly mutated EGFR tyrosine kinase domain (exons 18–21) was amplified by polymerase chain reaction using cDNA.20,21 Direct dideoxynucleotide sequencing was then used to analyze the amplified products. Fluorescence in Situ Hybridization Fluorescence in situ hybridization was performed using the LSI EGFR Spectrum orange/CEP7 Spectrum green probe (Abbott Laboratories, Abbott Park, IL) and LSI HER2 spectrum orange/CEP17 spectrum green probe (Abbott Laboratories) to assess the EGFR and HER2 gene copy number, respectively. At least 50 cells were assessed for each case by two pathologists and were classified in line with the published criteria as gene amplification (homogenously staining regions with C15 copies in C10 % of cells or a gene/chromosome ratio per cell of C2).11,22 Immunohistochemistry Briefly, paraffin-embedded tissue sections were analyzed for the protein expression of EGFR (EGF Receptor, D38B1, rabbit monoclonal antibody, 1:100 dilution; Cell Signaling Technology), p-EGFR (Tyr1068, Phospho-EGF Receptor Antibody, 1:50 dilution, Cell Signaling Technology, Danvers, MA), HER2 (Anti-HER2, 4B5, ready to use, Ventana), p-HER2 (Tyr1221/1222, Phospho-HER2/ErbB2, 6B12, rabbit monoclonal antibody, 1:100 dilution, Cell Signaling Technology), HER3 (specified designed, 1:2000 dilution, Cell Signaling Technology), p-HER3 (Tyr1289, PhosphoHER3/ErbB3, 21D3, rabbit monoclonal antibody, 1:150
EGFR Exon 20 Insertions in Lung Cancer
dilution, Cell Signaling Technology), p-Akt (Ser473, DAKO M3628, 1:50 dilution), and p-Erk (Thr202/Tyr204, 4376, 1:200 dilution, Cell Signaling Technology) after antigen retrieval. Immunostainings were evaluated by two observers in blind. Discrepancies were resolved through discussion. The tissue section immunoreactivity to all the antibodies was scored positive if more than 10 % of tumor cells showed staining of any intensity. Statistical Analysis We compared the clinicopathologic features of patients harboring EGFR exon 20 insertion mutations with patients harboring classic EGFR activating mutations (exon 19 deletions and L858R point mutation) using data from our previous study,22 which included 89 lung adenocarcinoma patients (28 exons, 19 deletions, and 26 L858R) who underwent surgery at our department from January 2008 to June 2009. The molecular characteristics in samples harboring EGFR exon 20 insertion mutations were compared with 35 patients with classic EGFR activating mutations (16 exons, 19 deletions, and 19 L858R) who underwent surgical resection at our department from January 2011 to August 2011. The association between two categorical variables was assessed by Pearson’s v2 test or by Fisher’s exact test. An independent sample t test was used to determine the correlation between a categorical variable and a continuous variable. Recurrence-free survival and overall survival was compared using the log-rank test. All statistical analyses were performed in the SPSS for Windows (version 16.0, Chicago, IL). P values were two-tailed for all the tests, and statistical significance was set as P \ 0.05. FIG. 1 Spectrum of EGFR exon 20 insertion mutations in lung adenocarcinoma
RESULTS Prevalence and Spectrum of EGFR Exon 20 Insertion Mutations in East Asian Lung Adenocarcinoma Patients A total of 662 (61.0 %) EGFR mutations were detected from 1,086 lung adenocarcinomas. Thirty-one patients testing positive for EGFR exon 20 insertion mutations were identified, accounting for 2.9 % of all lung adenocarcinoma cases and 4.7 % of lung adenocarcinoma patients harboring EGFR mutations. The spectrum of the 31 EGFR exon 20 insertion mutations is shown in Fig. 1. In the 31 samples with EGFR exon 20 insertions, we also genotyped other well-identified molecular alterations, including KRAS mutations, HER2 mutations, ALK fusions, BRAF mutations, PIK3CA mutations, and AKT1 mutations. There were 80 (7.4 %) KRAS mutations, 57 (5.2 %) ALK fusions, 25 (2.3 %) HER2 mutations, 21 (1.9 %) PIK3CA mutations, 14 (1.3 %) BRAF mutations, and 1 (0.1 %) AKT1 mutation. The EGFR exon 20 insertion mutations were mutually exclusive with other EGFR mutation types and with other well-identified oncogenic driver mutations. Clinicopathologic Features of Lung Adenocarcinomas Harboring EGFR Exon 20 Insertion Mutations There were 17 women and 14 men ranging in age from 34 to 77 years old (mean age 56.8 years old). Detailed clinicopathologic information is listed in Table 1. Patients with exon 20 insertion mutations were significantly younger than those with L858R point mutation
Y. Pan et al.
0.052
(P = 0.032). Morphologic spectrum of lung adenocarcinoma with exon 20 insertions did not differ significantly from the classic EGFR mutation cohorts. We included relapse-free and overall survival data of patients diagnosed before January 2011. Therefore, 17 patients with EGFR exon 20 insertion mutations and all the patients with the classic activating EGFR mutations were included for survival analysis (Fig. 2). Median follow-up was 39 months for the entire cohort and 28 months for patients with exon 20 insertion mutations. Relapse-free survival was significantly shorter for patients harboring EGFR exon 20 insertion mutations than it was for patients with exon 19 deletion mutations (P = 0.045). There was no significant difference in relapse-free survival between patients with exon 20 insertion mutations and L858R point mutation (P = 0.202). Overall survival was not significantly different among EGFR mutation subtypes [P = 0.989 for exon 20 insertion versus (vs) exon 19 deletion, and P = 0.905 for exon 20 insertion vs. L858R]. Molecular Associations of Lung Adenocarcinoma with EGFR Exon 20 Insertion Mutations
TABLE 1 Comparison of clinicopathologic characteristics between lung adenocarcinoma patients harboring EGFR exon 20 insertion mutations and those with classic activating mutations Variables
Exon 20 Exon 19 del (n = 28) L858R (n = 26) ins (n = 31) Pa Pb
Age (years) Mean
56.8
58.5
SD
10.0
10.2
61.9 0.521
6.8
0.648
12
0.032
Sex Female
17
17
14
11
Ever
9
9
Never
22
19
4
5
Male Smoking history
14 0.940
8 0.796
18
0.886
Differentiation Poor
Moderate/Well 27
23
9 0.723
17
Stage I–II
22
17
14
III–IV
9
11
0.406
12
0.182
Histologic subtypes AIS
0
0
ND
2
0.204
MIA
1
1
1.000
1
1.000
Lepidic
5
4
1.000
1
0.205
Papillary
8
6
0.693
10
0.306
Acinar Solid
15 1
13 4
0.880 0.180
7 5
0.097 0.083
0
1.000
0
1.000
Micropapillary 1
AIS adenocarcinoma in situ, del deletions, ins insertions, MIA minimally invasive adenocarcinoma, ND not done, SD stable disease a
Comparison between exon 20 insertions and exon 19 deletions
b
Comparison between exon 20 insertions and L858R
FIG. 2 Relapse-free survival a and overall survival b according to EGFR mutation subtypes. E19 del exon 19 deletions, E20 ins exon 20 insertions
Images of positive and negative controls of total or phosphorylated protein staining were shown in Supplemental Fig. S1. Protein expression of EGFR, HER2, and HER3 were positive in 27 (87.1 %), 2 (6.5 %), and 7 (22.6 %) of the cases, respectively (Table 2). The percentages of positive p-EGFR, p-HER2, and p-HER3 staining were all 3.2 % (1 of 31). Samples with EGFR exon 20 insertion mutations displayed a significantly lower percentage of positive total HER3 expression (22.6 vs. 51.4 %; P = 0.016) and p-EGFR expression (3.3 vs. 40 %; P \ 0.001) compared to those harboring classic activating EGFR mutations (EGFR exon 19 deletions and L858R).
a
b
Relapse-free survival
Overall survival
1.0
1.0
0.8
0.8
0.6
0.6
E19 del E20 ins L858R Censored
0.4
0.2
0.4
0.2
E20 ins vs. E19 del: P = 0.045
0
10
20
30
Months
40
E19 del E20 ins L858R Censored
50
60
0
10
20
30
Months
40
50
60
EGFR Exon 20 Insertions in Lung Cancer TABLE 2 Comparison of molecular features between lung adenocarcinomas harboring EGFR exon 20 insertion mutations and those with classic activating mutations Variables
Exon 20 ins
Classic mut
27
29
4
6
TABLE 3 Comparison of molecular characteristics between EGFR exon 20 insertion mutations within and after the C-helix Variables
Within the C-helix (n = 4)
After the C-helix (n = 27)
Positive
3
24
Negative
1
3
P
EGFR IHC Positive Negative p-EGFR Positive
1
14
Negative
30
21
EGFR IHC 0.739
p-EGFR \0.001
HER2 IHC Positive
2
1
Negative
29
34
1
3
Negative
30
32
Positive
7
18
Negative
24
17
1
5
30
30
0.616
Positive
21
12
Negative
10
23
0.016
21
33
Negative
10
2
0.202
No amplification
8
8
23
27
0.007
No amplification
1
3
30
32
1
3
26
Positive
0
1
Negative
4
26
0 4
7 20
Positive
0
1
Negative
4
26
Positive
4
17
Negative
0
10
Positive
1
20
Negative
3
7
Positive Negative
p-Erk 0.009
EGFR FISH 0.780
HER2 FISH Amplification
1
Negative
p-Akt
EGFR FISH Amplification
Positive
p-HER3
p-Erk Positive
26
HER3 IHC
p-HER3 Negative p-Akt
1
4
p-HER2
HER3 IHC
Positive
0
Negative HER2 IHC 0.597
p-HER2 Positive
Positive
Amplification
2
6
No amplification
2
21
0 4
1 26
HER2 FISH 0.616
Classic mut classic EGFR activating mutations including Exon 19 deletions and L858R; FISH fluorescence in situ hybridization, IHC immunohistochemistry, ins insertions, p phosphorylated
Amplification No amplification
FISH fluorescence in situ hybridization, IHC immunohistochemistry, p phosphorylated
EGFR Exon 20 Insertion Mutations Within the C-helix Positive p-Akt expression was observed in 21 (67.7 %) cases and was significantly more frequent in tumors harboring exon 20 insertion mutations (67.7 vs. 34.3 %; P = 0.007), whereas positive p-Erk expression was significantly less frequent in samples with exon 20 insertion mutations compared to those with classic activating EGFR mutations (67.7 vs. 94.3 %; P = 0.009). EGFR and HER2 gene amplification were found in 8 (25.8 %) and 1 (3.2 %) of the 31 samples, respectively. The frequency of gene amplification cases did not differ in EGFR mutation subtypes.
The 4 (12.9 %) EGFR exon 20 insertion mutations within the C-helix were all 763–764 insFQEA. They all had positive p-Akt expression, whereas only 1 of the 4 samples showed positive p-Erk expression (Table 3). Outcomes of Patients Treated with EGFR TKIs Three patients received EGFR TKIs after disease recurrence. Stable disease was observed in one patient with 769–770 insASV, and progressive disease was observed in two patients harboring 763–764 insFQEA and 770–771
Y. Pan et al.
insSVD, respectively. Detailed information was listed in Supplemental Table S1. DISCUSSION The clinicopathologic and molecular characteristics of lung adenocarcinoma patients harboring exon 20 insertion mutations remain poorly defined. Recently, two studies investigated the prevalence and clinicopathologic characteristics of NSCLC patients with EGFR exon 20 insertion mutations.23,24 However, the two studies mainly enrolled Caucasian patients, and molecular associations were not assessed. The only study specifically focused on East Asian NSCLC patients with EGFR exon 20 insertion mutations reported just seven patients.25 As far as we were concerned, our study represents the largest investigation for the prevalence, clinicopathologic features, and molecular correlations of EGFR exon 20 insertion mutations in East Asian lung adenocarcinoma patients. We found that EGFR exon 20 insertion mutations were present in 2.9 % of lung adenocarcinomas and 4.7 % of all EGFR mutations, and were mutually exclusive with other EGFR mutation types and other well-identified molecular alterations. The proportion of exon 20 insertion mutations in all EGFR mutations in our study was lower than the two large studies that mainly enrolled Caucasian patients (both of which reported a proportion of nearly 10 %),23,24 indicating that ethnic factors may affect the distribution of EGFR mutation subtypes, thus a large study specifically focusing on East Asian patients is necessary. The distinct characteristics of patients with exon 20 insertion mutations included younger age at diagnosis compared to those with L858R point mutations, suggesting their idiographic mechanism of carcinogenesis. More interestingly, we found that patients harboring exon 20 insertion mutations had significantly shorter relapse-free survival than those with exon 19 deletion mutations. As none of these patients received postoperative EGFR TKIs before disease relapse, our results suggest that exon 20 insertions may predict poor prognosis. ErbB receptors exist as monomers in the cell membrane. Ligand binding induces homodimerization and heterodimerization of the ErbB receptors, leading to the phosphorylation of tyrosine residues in their cytoplasmic domains with the consequent activation of downstream signaling pathways.13 HER3 is unique because it lacks a functional kinase and requires heterodimerization with other ErbBs to activate downstream kinases.26 An important point for a cell to be sensitive to EGFR inhibition is that the cell must rely on the EGFR and its downstream pathways for their survival. Engelman et al..16 revealed that HER3 is used to activate the PI3K/Akt pathway in
gefitinib-sensitive NSCLC cell lines and that HER3 expression strongly correlated with sensitivity to gefitinib. Fujimoto et al..27 showed that high expression of HER3 was associated with gefitinib sensitivity, regardless of EGFR mutational status, indicating that lung adenocarcinoma cells that rely on EGFR for survival constitutively activate the receptor through overexpression of EGFR dimeric partners (such as HER3) and through genetic mutations. In this study, the findings that patients harboring EGFR exon 20 insertion mutations showed lower expression of p-EGFR and HER3 than those with classic activating mutations suggested less dependence on EGFR for survival and therefore less sensitivity to EGFR TKIs. Our results showed that EGFR downstream pathways were still activated in samples harboring EGFR exon 20 insertion mutations, possibly through mechanisms other than EGFR tyrosine phosphorylation. However, compared to samples harboring classic activating mutations, those with exon 20 insertion mutations had a higher expression of p-Akt and a lower expression of p-Erk, suggesting different pathway activation patterns. We revealed that the EGFR exon 20 insertion mutations within the C-helix may account for approximately 10 % of all the exon 20 insertions. Yasuda et al..8 reported that the NSCLC harboring EGFR-A763 Y764insFQEA, which is located within the C-helix, achieved a partial response to erlotinib, and the cell line with this mutation was sensitive to nanomolar concentrations of erlotinib. This study showed that insertions within or after the C-helix may have different expression of downstream molecules, p-Akt and p-Erk, suggesting different patterns of downstream activation. Sufficient literature evidence has demonstrated the insensitivity of EGFR exon 20 insertion mutations to EGFR TKIs, which is why we did not suggest patients take TKIs after disease recurrence. Objective response was observed in none of the three patients who received EGFR TKIs, including one patient harboring insertion mutations within the C-helix (763–764 insFQEA). Although we could not draw any conclusion based on the small number of patients, these data could add to our knowledge on the efficacy of EGFR TKIs for patients with EGFR exon 20 insertion mutations. In conclusion, through analysis of more than 1,000 East Asian lung adenocarcinoma patients, we found that lung adenocarcinomas with EGFR exon 20 insertion mutations were present in a considerable proportion, displayed distinct clinicopathologic characteristics, had less dependence on EGFR molecularly, and had different pathway activation patterns compared to those harboring classic EGFR activating mutations. These results have implications to the development of therapeutic strategies targeting this significant cohort of lung cancer patients.
EGFR Exon 20 Insertions in Lung Cancer ACKNOWLEDGMENT This work was supported by Grants from the Key Construction Program of the National ‘‘985’’ Project (985IIIYFX0102), the National Natural Science Foundation of China (81172218, 81101760, and 81101761), and the Science and Technology Commission of Shanghai Municipality (Program of Shanghai Subject Chief Scientist) (12XD1402000). We thank the AstraZeneca Shanghai Innovation Center, Shanghai, China, for excellent technical support.
12. 13. 14.
15.
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ORIGINAL ARTICLE – TRANSLATIONAL RESEARCH AND BIOMARKERS
Prevalence, Clinicopathologic Characteristics, and Molecular Associations of EGFR Exon 20 Insertion Mutations in East Asian Patients with Lung Adenocarcinoma Yunjian Pan, MD1,2, Yang Zhang, MD1,2, Yuan Li, MD2,3, Haichuan Hu, MD1,2, Lei Wang, MD1,2, Hang Li, MD1,2, Rui Wang, MD1,2, Ting Ye, MD1,2, Xiaoyang Luo, MD1,2, Yiliang Zhang, MD1,2, Bin Li, MD1,2, Deng Cai, MD1,2, Lei Shen, MD2,3, Yihua Sun, MD1,2, and Haiquan Chen, MD1,2 Department of Thoracic Surgery, Fudan University Shanghai Cancer Center, Shanghai, China; 2Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China; 3Department of Pathology, Fudan University Shanghai Cancer Center, Shanghai, China 1
ABSTRACT Purpose. To define the prevalence, clinicopathologic characteristics and molecular associations of epidermal growth factor receptor (EGFR) exon 20 insertion mutations in East Asian lung adenocarcinoma patients. Methods. A total of 1,086 lung adenocarcinomas were sequenced for EGFR mutations. EGFR and HER2 copy number variations; total and phosphorylated (p) protein expression of ErbB family members including EGFR, HER2, and HER3; phosphorylated protein expression of downstream signaling molecules including Akt and Erk; and clinicopathologic features in lung adenocarcinomas with EGFR exon 20 insertion mutations were all investigated. Results. EGFR exon 20 insertion mutations were present in 2.9 % of lung adenocarcinomas and 4.7 % of all the EGFR mutations. Compared to those with classic activating EGFR mutations, lung adenocarcinomas with exon 20
insertion mutations were characterized by significantly younger age at diagnosis (P = 0.032 for exon 20 insertions vs. L858R) and shorter relapse-free survival [P = 0.045 for exon 20 insertions versus (vs) exon 19 deletions]. Molecularly, samples harboring exon 20 insertion mutations had lower expression of phosphorylated (p)-EGFR (P \ 0.001) and HER3 (P = 0.016). In addition, higher expression of p-Akt (P = 0.007) and lower expression of p-Erk (P = 0.009) were observed in tumors with exon 20 insertion mutations. Conclusions. Lung adenocarcinomas with EGFR exon 20 insertion mutations were present in a substantial proportion. This subset showed distinct clinicopathologic features, less dependence on EGFR molecularly, and different pathway activation patterns compared to those with classic EGFR activating mutations.
INTRODUCTION
Yunjian Pan and Yang Zhang have contributed equally to this work and should both be considered first co-authors.
Electronic supplementary material The online version of this article (doi:10.1245/s10434-013-3452-1) contains supplementary material, which is available to authorized users. Ó Society of Surgical Oncology 2014 First Received: 18 August 2013 Y. Sun, MD e-mail: [email protected] H. Chen, MD e-mail: [email protected]
Lung cancer is the most prevalent cancer in male patients and the fourth most commonly diagnosed cancer among female patients.1 Non-small cell lung cancer (NSCLC) is now a disease that can be classified into clinically relevant molecular subgroups, each with distinct clinicopathologic features and potential for targeted therapies. Among these molecular subsets, the identification of epidermal growth factor receptor (EGFR) tyrosine kinase domain mutations that predict sensitivity to tyrosine kinase inhibitors (TKIs) is the most prominent event.2–4 Patients with EGFR mutations, however, are also a heterogeneous group with different sensitivity to TKIs. Although the classic exon 19 deletions and L858R point
Y. Pan et al.
mutation in exon 21 usually confer sensitivity to reversible TKIs, gefitinib, and erlotinib, most exon 20 insertion mutations reported to date are associated with resistance to TKIs.5–7 Exon 20 insertion mutations can also be classified into two subgroups by aminoacid position: insertions that affect the amino acids after the C-helix tyrosine kinase domain (Ala767 to Cys775) of the EGFR and within the C-helix (Glu762 to Met766) of the EGFR. The former account for the majority (90 %) of all reported exon 20 insertions and show de novo resistance to clinically achievable doses of gefitinib and erlotinib,5 although the latter accounts for 4 % and may be labeled EGFR TKIsensitive mutations.8 The mechanisms underlying the differential sensitivity to EGFR TKIs between EGFR exon 20 insertion mutations and classic activating mutations are largely unexplored. Despite EGFR tyrosine kinase domain mutations, other EGFR gene status including increased copy number and protein expression were reported to predict response to EGFR TKIs.9–11 EGFR (ErbB1) is a member of the ErbB family. Other ErbB family members including HER2 (ErbB2) and HER3 (ErbB3) are also important in the development and progression of human cancer.12 Ligand binding induces homodimerization and heterodimerization of the ErbB receptors, leading to the autophosphorylation of their cytoplasmic domains and the consequent activation of downstream proteins.13 Accordingly, some studies reported a correlation between the expression of the HER2 and HER3, and the therapeutic efficacy of EGFR TKIs.14–16 Moreover, because EGFR activity is mediated by downstream signaling cascade, including the PI3 K/Akt and Ras/Raf/Erk pathways, the possible roles of phosphorylated (p)-Akt and p-Erk as predictive markers have also been reported.17,18 We hypothesized that there may be differential expression of these molecular predictors between EGFR exon 20 insertion mutations and classic activating mutations, which may provide some explanation to the differential sensitivity to EGFR TKIs. We, therefore, sequenced more than 1,000 lung adenocarcinomas to investigate the clinicopathologic features of patients harboring EGFR exon 20 insertion mutations. We also explored the status of other reported predictive molecular markers for EGFR TKIs in samples harboring EGFR exon 20 insertion mutations. MATERIALS AND METHODS Patients and Samples From August 2007 to October 2012, we consecutively collected tumor tissues from lung adenocarcinoma patients who underwent surgical resection at the Department of Thoracic Surgery, Fudan University Shanghai Cancer
Center, Shanghai, China. Eligible criteria included pathologic confirmation of lung adenocarcinoma, each sample containing a minimum of 50 % of tumor cells and sufficient tissue for comprehensive mutational analyses. Patients who received neoadjuvant therapies were excluded. Clinicopathologic variables collected for analyses included age at diagnosis, gender, smoking history, tumor differentiation, pathologic TNM stage, and histologic subtypes in line with the new International Association for the Study of Lung Cancer, the American Thoracic Society, and the European Respiratory Society international multidisciplinary classification of lung adenocarcinoma.19 Disease recurrence and survival were recorded according to follow-up clinic visits or telephone calls. Our institutional review board approved this study, and written informed consent was obtained from all patients. EGFR Mutational Analysis RNA was extracted according to standard protocol from frozen tumor specimens. Total RNA samples were then reverse transcribed into complementary DNA. The most commonly mutated EGFR tyrosine kinase domain (exons 18–21) was amplified by polymerase chain reaction using cDNA.20,21 Direct dideoxynucleotide sequencing was then used to analyze the amplified products. Fluorescence in Situ Hybridization Fluorescence in situ hybridization was performed using the LSI EGFR Spectrum orange/CEP7 Spectrum green probe (Abbott Laboratories, Abbott Park, IL) and LSI HER2 spectrum orange/CEP17 spectrum green probe (Abbott Laboratories) to assess the EGFR and HER2 gene copy number, respectively. At least 50 cells were assessed for each case by two pathologists and were classified in line with the published criteria as gene amplification (homogenously staining regions with C15 copies in C10 % of cells or a gene/chromosome ratio per cell of C2).11,22 Immunohistochemistry Briefly, paraffin-embedded tissue sections were analyzed for the protein expression of EGFR (EGF Receptor, D38B1, rabbit monoclonal antibody, 1:100 dilution; Cell Signaling Technology), p-EGFR (Tyr1068, Phospho-EGF Receptor Antibody, 1:50 dilution, Cell Signaling Technology, Danvers, MA), HER2 (Anti-HER2, 4B5, ready to use, Ventana), p-HER2 (Tyr1221/1222, Phospho-HER2/ErbB2, 6B12, rabbit monoclonal antibody, 1:100 dilution, Cell Signaling Technology), HER3 (specified designed, 1:2000 dilution, Cell Signaling Technology), p-HER3 (Tyr1289, PhosphoHER3/ErbB3, 21D3, rabbit monoclonal antibody, 1:150
EGFR Exon 20 Insertions in Lung Cancer
dilution, Cell Signaling Technology), p-Akt (Ser473, DAKO M3628, 1:50 dilution), and p-Erk (Thr202/Tyr204, 4376, 1:200 dilution, Cell Signaling Technology) after antigen retrieval. Immunostainings were evaluated by two observers in blind. Discrepancies were resolved through discussion. The tissue section immunoreactivity to all the antibodies was scored positive if more than 10 % of tumor cells showed staining of any intensity. Statistical Analysis We compared the clinicopathologic features of patients harboring EGFR exon 20 insertion mutations with patients harboring classic EGFR activating mutations (exon 19 deletions and L858R point mutation) using data from our previous study,22 which included 89 lung adenocarcinoma patients (28 exons, 19 deletions, and 26 L858R) who underwent surgery at our department from January 2008 to June 2009. The molecular characteristics in samples harboring EGFR exon 20 insertion mutations were compared with 35 patients with classic EGFR activating mutations (16 exons, 19 deletions, and 19 L858R) who underwent surgical resection at our department from January 2011 to August 2011. The association between two categorical variables was assessed by Pearson’s v2 test or by Fisher’s exact test. An independent sample t test was used to determine the correlation between a categorical variable and a continuous variable. Recurrence-free survival and overall survival was compared using the log-rank test. All statistical analyses were performed in the SPSS for Windows (version 16.0, Chicago, IL). P values were two-tailed for all the tests, and statistical significance was set as P \ 0.05. FIG. 1 Spectrum of EGFR exon 20 insertion mutations in lung adenocarcinoma
RESULTS Prevalence and Spectrum of EGFR Exon 20 Insertion Mutations in East Asian Lung Adenocarcinoma Patients A total of 662 (61.0 %) EGFR mutations were detected from 1,086 lung adenocarcinomas. Thirty-one patients testing positive for EGFR exon 20 insertion mutations were identified, accounting for 2.9 % of all lung adenocarcinoma cases and 4.7 % of lung adenocarcinoma patients harboring EGFR mutations. The spectrum of the 31 EGFR exon 20 insertion mutations is shown in Fig. 1. In the 31 samples with EGFR exon 20 insertions, we also genotyped other well-identified molecular alterations, including KRAS mutations, HER2 mutations, ALK fusions, BRAF mutations, PIK3CA mutations, and AKT1 mutations. There were 80 (7.4 %) KRAS mutations, 57 (5.2 %) ALK fusions, 25 (2.3 %) HER2 mutations, 21 (1.9 %) PIK3CA mutations, 14 (1.3 %) BRAF mutations, and 1 (0.1 %) AKT1 mutation. The EGFR exon 20 insertion mutations were mutually exclusive with other EGFR mutation types and with other well-identified oncogenic driver mutations. Clinicopathologic Features of Lung Adenocarcinomas Harboring EGFR Exon 20 Insertion Mutations There were 17 women and 14 men ranging in age from 34 to 77 years old (mean age 56.8 years old). Detailed clinicopathologic information is listed in Table 1. Patients with exon 20 insertion mutations were significantly younger than those with L858R point mutation
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0.052
(P = 0.032). Morphologic spectrum of lung adenocarcinoma with exon 20 insertions did not differ significantly from the classic EGFR mutation cohorts. We included relapse-free and overall survival data of patients diagnosed before January 2011. Therefore, 17 patients with EGFR exon 20 insertion mutations and all the patients with the classic activating EGFR mutations were included for survival analysis (Fig. 2). Median follow-up was 39 months for the entire cohort and 28 months for patients with exon 20 insertion mutations. Relapse-free survival was significantly shorter for patients harboring EGFR exon 20 insertion mutations than it was for patients with exon 19 deletion mutations (P = 0.045). There was no significant difference in relapse-free survival between patients with exon 20 insertion mutations and L858R point mutation (P = 0.202). Overall survival was not significantly different among EGFR mutation subtypes [P = 0.989 for exon 20 insertion versus (vs) exon 19 deletion, and P = 0.905 for exon 20 insertion vs. L858R]. Molecular Associations of Lung Adenocarcinoma with EGFR Exon 20 Insertion Mutations
TABLE 1 Comparison of clinicopathologic characteristics between lung adenocarcinoma patients harboring EGFR exon 20 insertion mutations and those with classic activating mutations Variables
Exon 20 Exon 19 del (n = 28) L858R (n = 26) ins (n = 31) Pa Pb
Age (years) Mean
56.8
58.5
SD
10.0
10.2
61.9 0.521
6.8
0.648
12
0.032
Sex Female
17
17
14
11
Ever
9
9
Never
22
19
4
5
Male Smoking history
14 0.940
8 0.796
18
0.886
Differentiation Poor
Moderate/Well 27
23
9 0.723
17
Stage I–II
22
17
14
III–IV
9
11
0.406
12
0.182
Histologic subtypes AIS
0
0
ND
2
0.204
MIA
1
1
1.000
1
1.000
Lepidic
5
4
1.000
1
0.205
Papillary
8
6
0.693
10
0.306
Acinar Solid
15 1
13 4
0.880 0.180
7 5
0.097 0.083
0
1.000
0
1.000
Micropapillary 1
AIS adenocarcinoma in situ, del deletions, ins insertions, MIA minimally invasive adenocarcinoma, ND not done, SD stable disease a
Comparison between exon 20 insertions and exon 19 deletions
b
Comparison between exon 20 insertions and L858R
FIG. 2 Relapse-free survival a and overall survival b according to EGFR mutation subtypes. E19 del exon 19 deletions, E20 ins exon 20 insertions
Images of positive and negative controls of total or phosphorylated protein staining were shown in Supplemental Fig. S1. Protein expression of EGFR, HER2, and HER3 were positive in 27 (87.1 %), 2 (6.5 %), and 7 (22.6 %) of the cases, respectively (Table 2). The percentages of positive p-EGFR, p-HER2, and p-HER3 staining were all 3.2 % (1 of 31). Samples with EGFR exon 20 insertion mutations displayed a significantly lower percentage of positive total HER3 expression (22.6 vs. 51.4 %; P = 0.016) and p-EGFR expression (3.3 vs. 40 %; P \ 0.001) compared to those harboring classic activating EGFR mutations (EGFR exon 19 deletions and L858R).
a
b
Relapse-free survival
Overall survival
1.0
1.0
0.8
0.8
0.6
0.6
E19 del E20 ins L858R Censored
0.4
0.2
0.4
0.2
E20 ins vs. E19 del: P = 0.045
0
10
20
30
Months
40
E19 del E20 ins L858R Censored
50
60
0
10
20
30
Months
40
50
60
EGFR Exon 20 Insertions in Lung Cancer TABLE 2 Comparison of molecular features between lung adenocarcinomas harboring EGFR exon 20 insertion mutations and those with classic activating mutations Variables
Exon 20 ins
Classic mut
27
29
4
6
TABLE 3 Comparison of molecular characteristics between EGFR exon 20 insertion mutations within and after the C-helix Variables
Within the C-helix (n = 4)
After the C-helix (n = 27)
Positive
3
24
Negative
1
3
P
EGFR IHC Positive Negative p-EGFR Positive
1
14
Negative
30
21
EGFR IHC 0.739
p-EGFR \0.001
HER2 IHC Positive
2
1
Negative
29
34
1
3
Negative
30
32
Positive
7
18
Negative
24
17
1
5
30
30
0.616
Positive
21
12
Negative
10
23
0.016
21
33
Negative
10
2
0.202
No amplification
8
8
23
27
0.007
No amplification
1
3
30
32
1
3
26
Positive
0
1
Negative
4
26
0 4
7 20
Positive
0
1
Negative
4
26
Positive
4
17
Negative
0
10
Positive
1
20
Negative
3
7
Positive Negative
p-Erk 0.009
EGFR FISH 0.780
HER2 FISH Amplification
1
Negative
p-Akt
EGFR FISH Amplification
Positive
p-HER3
p-Erk Positive
26
HER3 IHC
p-HER3 Negative p-Akt
1
4
p-HER2
HER3 IHC
Positive
0
Negative HER2 IHC 0.597
p-HER2 Positive
Positive
Amplification
2
6
No amplification
2
21
0 4
1 26
HER2 FISH 0.616
Classic mut classic EGFR activating mutations including Exon 19 deletions and L858R; FISH fluorescence in situ hybridization, IHC immunohistochemistry, ins insertions, p phosphorylated
Amplification No amplification
FISH fluorescence in situ hybridization, IHC immunohistochemistry, p phosphorylated
EGFR Exon 20 Insertion Mutations Within the C-helix Positive p-Akt expression was observed in 21 (67.7 %) cases and was significantly more frequent in tumors harboring exon 20 insertion mutations (67.7 vs. 34.3 %; P = 0.007), whereas positive p-Erk expression was significantly less frequent in samples with exon 20 insertion mutations compared to those with classic activating EGFR mutations (67.7 vs. 94.3 %; P = 0.009). EGFR and HER2 gene amplification were found in 8 (25.8 %) and 1 (3.2 %) of the 31 samples, respectively. The frequency of gene amplification cases did not differ in EGFR mutation subtypes.
The 4 (12.9 %) EGFR exon 20 insertion mutations within the C-helix were all 763–764 insFQEA. They all had positive p-Akt expression, whereas only 1 of the 4 samples showed positive p-Erk expression (Table 3). Outcomes of Patients Treated with EGFR TKIs Three patients received EGFR TKIs after disease recurrence. Stable disease was observed in one patient with 769–770 insASV, and progressive disease was observed in two patients harboring 763–764 insFQEA and 770–771
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insSVD, respectively. Detailed information was listed in Supplemental Table S1. DISCUSSION The clinicopathologic and molecular characteristics of lung adenocarcinoma patients harboring exon 20 insertion mutations remain poorly defined. Recently, two studies investigated the prevalence and clinicopathologic characteristics of NSCLC patients with EGFR exon 20 insertion mutations.23,24 However, the two studies mainly enrolled Caucasian patients, and molecular associations were not assessed. The only study specifically focused on East Asian NSCLC patients with EGFR exon 20 insertion mutations reported just seven patients.25 As far as we were concerned, our study represents the largest investigation for the prevalence, clinicopathologic features, and molecular correlations of EGFR exon 20 insertion mutations in East Asian lung adenocarcinoma patients. We found that EGFR exon 20 insertion mutations were present in 2.9 % of lung adenocarcinomas and 4.7 % of all EGFR mutations, and were mutually exclusive with other EGFR mutation types and other well-identified molecular alterations. The proportion of exon 20 insertion mutations in all EGFR mutations in our study was lower than the two large studies that mainly enrolled Caucasian patients (both of which reported a proportion of nearly 10 %),23,24 indicating that ethnic factors may affect the distribution of EGFR mutation subtypes, thus a large study specifically focusing on East Asian patients is necessary. The distinct characteristics of patients with exon 20 insertion mutations included younger age at diagnosis compared to those with L858R point mutations, suggesting their idiographic mechanism of carcinogenesis. More interestingly, we found that patients harboring exon 20 insertion mutations had significantly shorter relapse-free survival than those with exon 19 deletion mutations. As none of these patients received postoperative EGFR TKIs before disease relapse, our results suggest that exon 20 insertions may predict poor prognosis. ErbB receptors exist as monomers in the cell membrane. Ligand binding induces homodimerization and heterodimerization of the ErbB receptors, leading to the phosphorylation of tyrosine residues in their cytoplasmic domains with the consequent activation of downstream signaling pathways.13 HER3 is unique because it lacks a functional kinase and requires heterodimerization with other ErbBs to activate downstream kinases.26 An important point for a cell to be sensitive to EGFR inhibition is that the cell must rely on the EGFR and its downstream pathways for their survival. Engelman et al..16 revealed that HER3 is used to activate the PI3K/Akt pathway in
gefitinib-sensitive NSCLC cell lines and that HER3 expression strongly correlated with sensitivity to gefitinib. Fujimoto et al..27 showed that high expression of HER3 was associated with gefitinib sensitivity, regardless of EGFR mutational status, indicating that lung adenocarcinoma cells that rely on EGFR for survival constitutively activate the receptor through overexpression of EGFR dimeric partners (such as HER3) and through genetic mutations. In this study, the findings that patients harboring EGFR exon 20 insertion mutations showed lower expression of p-EGFR and HER3 than those with classic activating mutations suggested less dependence on EGFR for survival and therefore less sensitivity to EGFR TKIs. Our results showed that EGFR downstream pathways were still activated in samples harboring EGFR exon 20 insertion mutations, possibly through mechanisms other than EGFR tyrosine phosphorylation. However, compared to samples harboring classic activating mutations, those with exon 20 insertion mutations had a higher expression of p-Akt and a lower expression of p-Erk, suggesting different pathway activation patterns. We revealed that the EGFR exon 20 insertion mutations within the C-helix may account for approximately 10 % of all the exon 20 insertions. Yasuda et al..8 reported that the NSCLC harboring EGFR-A763 Y764insFQEA, which is located within the C-helix, achieved a partial response to erlotinib, and the cell line with this mutation was sensitive to nanomolar concentrations of erlotinib. This study showed that insertions within or after the C-helix may have different expression of downstream molecules, p-Akt and p-Erk, suggesting different patterns of downstream activation. Sufficient literature evidence has demonstrated the insensitivity of EGFR exon 20 insertion mutations to EGFR TKIs, which is why we did not suggest patients take TKIs after disease recurrence. Objective response was observed in none of the three patients who received EGFR TKIs, including one patient harboring insertion mutations within the C-helix (763–764 insFQEA). Although we could not draw any conclusion based on the small number of patients, these data could add to our knowledge on the efficacy of EGFR TKIs for patients with EGFR exon 20 insertion mutations. In conclusion, through analysis of more than 1,000 East Asian lung adenocarcinoma patients, we found that lung adenocarcinomas with EGFR exon 20 insertion mutations were present in a considerable proportion, displayed distinct clinicopathologic characteristics, had less dependence on EGFR molecularly, and had different pathway activation patterns compared to those harboring classic EGFR activating mutations. These results have implications to the development of therapeutic strategies targeting this significant cohort of lung cancer patients.
EGFR Exon 20 Insertions in Lung Cancer ACKNOWLEDGMENT This work was supported by Grants from the Key Construction Program of the National ‘‘985’’ Project (985IIIYFX0102), the National Natural Science Foundation of China (81172218, 81101760, and 81101761), and the Science and Technology Commission of Shanghai Municipality (Program of Shanghai Subject Chief Scientist) (12XD1402000). We thank the AstraZeneca Shanghai Innovation Center, Shanghai, China, for excellent technical support.
12. 13. 14.
15.
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